Header image with fluorescence microscopy image showing cells with supernumerary centrosomes

Geoffrey A. Charters MSc (Hons) PhD (Pathology)

Molecular oncopathologist

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Doctor of Philosophy in Pathology1998 - 2007; awarded 2008

Institution:The University of Auckland, New Zealand
Thesis:Human metastatic melanoma in vitro: ploidy, centrosomal integrity, serum dependency, chromosome 9p21 genetic status, tumour-suppressor gene expression.
Abstract:

Metastatic melanoma cells frequently have flaws in the proliferative control mediated by the retinoblastoma-associated protein (pRB), normally operative at the G1-S cell-cycle phase transition. Functional and molecular aspects of this were investigated in seventeen human metastatic melanoma cell-lines (NZMs).

Flow-cytometry revealed aneuploidy and heteroploidy in many NZMs that were unstable over time. This plasticity may contribute to melanoma's therapeutic resistance. A potential cause of this, dysregulation of centrosomal numerical control, was demonstrated by immunofluorescence microscopy, apparently for the first time in melanoma cell-lines.

Pericentrin was found to accumulate in nucleolar reservoirs, previously unreported, and release from these on nuclear envelope breakdown may trigger mitotic spindle formation. Dysregulation of pericentrin may be particularly important in melanoma, and could represent a therapeutic target.

To test the integrity of pRB-mediated proliferative regulation, NZM cells were grown under conditions of serum deprivation that in normal cells would cause arrest via pRB. Cell-cycle phase analysis revealed three classes of response: accumulation in G1, accumulation in G2 or mitosis, or stimulation to enter S phase. G1 arrest indicates that proliferative regulation in response to serum deprivation may be normal in some melanomas, implying that the pRB subsystem may not be the sole regulator of this, or that it is not defective in all melanomas.

Using PCR deletion analysis, an investigation was undertaken into the integrity of the tandem 9p21 CDKN2A and CDKN2B genes that encode tumour-suppressors implicated in melanoma tumorigenesis. Homozygous deletions affecting only CDKN2A were found in two cell-lines, and affecting both genes in six. In the case of NZM7, where different sub-clones exist, heterogeneity was found by microsatellite analysis. DNA sequencing revealed a known CDKN2A G500C polymorphism in the NZM7 group, also heterogeneous among sub-clones. A CDKN2B G411A polymorphism was found in NZM14, but it is predicted not to affect the amino acid sequence of the encoded protein.

Protein analysis revealed that all NZMs express pRB, but in some, this was in the inhibitory unphosphorylated state, despite their being proliferative. This correlated with strong p16 expression and known BRAF mutations, suggesting that proliferation of BRAF mutants may require compromised function of the pRB subsystem.

Master of Science in Biological Sciences, Second Class Honours, Division 11995 - 1997; awarded 2008

Institution:The University of Auckland, New Zealand
Papers:Advanced Cellular and Molecular Biology for Biomedical Research, Molecular Genetics, and Cancer Biology
Thesis:Aspects of growth regulation in cultured human melanoma cells
Abstract:

The work at hand describes investigations undertaken between October 1995 and February 1997 at the Cancer Research Laboratory of the New Zealand Cancer Society (Auckland Division). The intention was to explore the different sensitivities of the human melanoma cell-lines NZM4 and NZM7 to growth inhibition by the cytokine TGF-β.

Several TGF-β resistance mechanisms were considered. One in particular became prominent. In TGF-β-treated mink lung epithelial cells cyclin-dependent kinase four (cdk4) is translationally down-regulated in a p53-dependent manner. In unrelated work, evidence was found for a pathway leading to G1 arrest after irradiation that depended upon p53, but not p21CDKN1, the established route. Together, these reports suggested that the same mechanism may be involved in both cases.

Flow cytometric analysis of the G1 cdk4 content of cultures revealed no evidence for down-regulation in response to TGF-β, etoposide or bleomycin. Surprisingly, indications of dose-dependent down-regulation were found in NZM7 after irradiation but this was not associated with the proportion of G0-G1 cells and cannot be causal.

Strong evidence was found of a minimum cdk4 concentration threshold that must be surpassed before entrance to S phase implying that cdk4 is rate-limiting for cycle progression in these cells. As no increase in cdk4 concentration was seen during S phase, the implication is that immediately after mitosis insufficient cdk4 will be present to sponsor immediate re-entry to S phase and consequently the occurrence of a G1 phase is assured.

This minimum level was found to persist throughout S phase, an observation that opposes the hypothesis that cyclin D-cdk4 is required only for S phase entry. A correlation was found between reduction in cdk4 levels and the onset of chromatin condensation before mitosis and there were indications that this may be a prerequisite for such condensation but the data were inconclusive.

In all, a great deal was learnt about laboratory procedure particularly concerning the maintenance of human cell-lines in tissue culture and techniques of flow cytometry.

Several novel observations are reported: down-regulation of cdk4 in response to irradiation, maintenance of a minimum cdk4 level throughout S phase, and the association between cdk4 down-regulation and chromatin condensation.

Certificate in Winemaking1993 - 1994; awarded 1994

Institution:Hawke's Bay Polytechnic, Napier, New Zealand

Bachelor of Science1978 - 1980; awarded 1984

Institution:The University of Auckland, New Zealand
Majors:Computer Science and Cell Biology